Promoter methylation of IGFBP-3 and p53 expression in ovarian endometrioid carcinoma

Abstract

Background: Insulin-like growth factor binding protein (IGFBP-3) is an antiproliferative, pro-apoptotic and invasion suppressor protein which is transcriptionally regulated by p53. Promoter methylation has been linked to gene silencing and cancer progression. We studied the correlation between IGFBP-3 and p53 expression as well as IGFBP-3 promoter methylation in ovarian endometrioid carcinoma (OEC) by immunohistochemical staining and quantitative methylation-specific PCR (qMSP). Additionally, we assessed the molecular regulatory mechanism of wild type (wt) p53 on IGFBP-3 expression using two subclones of OEC, the OVTW59-P0 (low invasive) and P4 (high invasive) sublines.

Results: In 60 cases of OEC, 40.0% showed lower IGFBP-3 expression which was significantly correlated with higher IGFBP-3 promoter methylation. p53 overexpression was detected in 35.0% of OEC and was unrelated to clinical outcomes and IGFBP-3. By Kaplan-Meier analysis, patients with lower IGFBP-3, higher IGFBP-3 promoter methylation, and normal p53 were associated most significantly with lower survival rates. In OEC cell line, IGFBP-3 expression was correlated with IGFBP-3 promoter methylation. IGFBP-3 expression was restored after treatment with a DNA methy-transferase inhibitors (5-aza-deoxycytidine) and suppressed by a p53 inhibitor (pifithrin-alpha). The putative p53 regulatory sites on the promoter of IGFBP-3 were identified at -210, -206, -183 and -179 bases upstream of the transcription start site. Directed mutagenesis at these sites quantitatively reduced the transcription activity of IGFBP-3.

Conclusion: Our data suggests that IGFBP-3 silencing through IGFBP-3 promoter methylation in the absence of p53 overexpression is associated with cancer progression. These results support a potential role of IGFBP-3 methylation in the carcinogenesis of OEC.

Figure 1

Immunohistochemical staining. High IGFBP-3 expression and normal p53 in a case of grade 1 OEC (a and b); and low IGFBP-3 expression and overexpression of p53 in a case of grade 3 OEC (c and d) (a - d, H&E, × 200).

Figure 2

Kaplan Meier survival estimate of progression-free survival (PFS) and overall survival (OS) for OEC patients classified by (a) IGFBP-3 expression, (b) IGFBP-3 promoter methylation status and (c) p53 expression. (d) Survival estimate for patients with normal p53, classified according to IGFBP-3 expressions and IGFBP-3 promoter methylation status. --- indicates patients with high IGFBP-3 and IGFBP-3 promoter methylation ≤ 3%; --- indicates patients with unparallel IGFBP-3 and IGFBP-3 promoter methylation, i.e. either high IGFBP-3 and IGFBP-3 promoter methylation >; 3% or low IGFBP-3 and IGFBP-3 promoter methylation ≤ 3%; --·-indicates patients with low IGFBP-3 and IGFBP-3 promoter methylation > 3%. Logrank test was used to compare survival distributions.

Figure 3

Aberrant promoter methylation and transcriptional regulation of p53 at IGFBP-3. (a) MSP shows IGFBP-3 promoter methylation in OEC cell lines. Upper panel: schematic diagram showing CpG islands in the IGFBP-3 promoter and the location of primers used (M, methylated; U, unmethylated). Arabic numbers indicate the location of nucleotides relative to the transcription start site (TSS) (+1). Vertical lines represent the position of the CpG dinucleotides. Lower panel: MSP in P0 and P4 sublines. U indicates unmethylated (158 bp) and M indicates methylated (129 bp) PCR products. The SssI treated A549 (lanes 1 and 2) and H1299 (lanes 4 and 5) were used as methylated controls, A549 as unmethylated control (lanes 6 and 7), and Genomic DNA without bisulfite conversion as negative control (lanes 12 and 13). (b) Real-time PCR analysis of IGFBP-3 mRNA showing restoration of IGFBP-3 expression in P4. P0 and P4 cell lines were treated with 0, 0.1, 0.5 or 2 μM of 5-aza-dC for 8 days. Culture media was changed to serum free media for 24 hours before IGFBP-3 analysis. Lower panel shows Western blotting corresponding to IGFBP-3 expressions in P0 and P4 culture media after 5-aza-dC treatment. Similar amount of culture media from equal number of cells were loaded in each lane. (c) Luciferase activity after transfection with wild type IGFBP-3 promoter (-253 ~+61)-pGL3 luciferase reporter construct (pGL3-wt IGFBP-3). Luciferase activity was normalized against renilla activity. Transfection with pGL3-basic vector (pGL3) was used as negative control. Transfections were carried out in triplicate and were done in three independent experiments.

Figure 4

Identification of methylation sites at p53 binding sequence of IGFBP-3 promoter. (a) BSP in P0 and P4. Forty CpG sites were shown and each circle represents a CpG dinucleotide. Open circle represents non-methylated CpG dinucleotude and black circle represents methylated CpG dinucleotide. The enclosed boxes represent CpG dinucleotide located in the p53 consensus binding site. Seven clones were sequenced in each cell line. (b) The methylation frequency in P0 and P4 from -210 to -179 regions of IGFBP-3 promoter. (c) Schematic diagram of designed constructs for site-directed mutagenesis assay. The wild type construct contains IGFBP-3 promoter region from --253 ~+61. The mutant sequences carry the same region but with point mutations: (-210, -206) represent as Mut A constructs, and (-183, -179) represent as Mut B constructs. Mut A+B construct contains all four nucleotide mutation. (d) Luciferase activities of 293T and P4 transfected with wild/mutant types of IGFBP-3 promoter constructs. Luciferase activity was normalized against renilla activity. Transfection with pGL3-basic vector (pGL3) was used as negative control. Transfections were carried out in triplicate and were done in at least three independent experiments.